論文 - 佐々木 康幸
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Role of DevR phosphorylation in nitric oxide homeostasis and signaling of Streptomyces coelicolor A3(2) M145 査読あり 国際誌
Sota Honm, Shinsaku Ito, Shunsuke Yajima, Yasuyuki Sasaki
FEMS Microbiol Lett . 2023年
担当区分：責任著者 記述言語：英語
Our previous studies revealed that a two-component system (TCS), DevS, and DevR, regulate both nitric oxide (NO) signaling and NO homeostasis in the actinobacterium Streptomyces coelicolor A3(2) M145, suggesting a reasonable system for NO-dependent metabolism. In this study, sequence alignment of DevR and DevR homologs found Asp66 (D66) and Thr196 (T196) as predicted phosphorylation sites of DevR. Phos-tag gel electrophoretic mobility shift assay suggested that D66 and T196 are involved in the phosphorylation of DevR. The respective point mutations of D66 and T196 significantly decreased the transcriptional activity of DevR, which affected nitrite production and aerial mycelium formation. These results suggested that both D66 and T196 of DevR are important for the regulation of NO homeostasis and signaling in S. coelicolor A3(2) M145.
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Synthesis of Carlactone Derivatives to Develop a Novel Inhibitor of Strigolactone Biosynthesis 査読あり 国際誌
Kojiro Kawada, Tatsuo Saito, Satoshi Onoda, Takuma Inayama, Ikuo Takahashi, Yoshiya Seto, Takahito Nomura, Yasuyuki Sasaki, Tadao Asami, Shunsuke Yajima, Shinsaku Ito
ACS Omega 8 ( 15 ) 2023年
Strigolactones (SLs), phytohormones that inhibit shoot branching in plants, promote the germination of root-parasitic plants, such as Striga spp. and Orobanche spp., which drastically reduces the crop yield. Therefore, reducing SL production via chemical treatment may increase the crop yield. To design specific inhibitors, it is valid to utilize the substrate structure of the target proteins as lead compounds. In this study, we focused on Os900, a rice enzyme that oxidizes the SL precursor carlactone (CL) to 4-deoxyorobanchol (4DO), and synthesized 10 CL derivatives. The effects of the synthesized CL derivatives on SL biosynthesis were evaluated by the Os900 enzyme assay in vitro and by measuring 4DO levels in rice root exudates. We identified some CL derivatives that inhibited SL biosynthesis in vitro and in vivo.
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Fenoxycarb, a carbamate insect growth regulator, inhibits brassinosteroid action 査読あり
Ito Shinsaku, Kawada Kojiro, Saeki Yasumasa, Nakano Takeshi, Sasaki Yasuyuki, Yajima Shunsuke
Journal of Pesticide Science advpub ( 0 ) 2023年
記述言語：英語 掲載種別：研究論文（学術雑誌） 出版者・発行元：Pesticide Science Society of Japan
<p>Brassinosteroids (BRs) are steroid hormones that regulate plant growth, development, and stress resistance. In this study, we evaluated the effect of agrochemicals on dark-induced hypocotyl elongation, which is regulated by BRs, to identify novel chemicals that regulate BR action. We found that the juvenile hormone agonist fenoxycarb inhibited dark-induced hypocotyl elongation in <i>Arabidopsis</i>. Treatment with the same class of juvenile hormone agonist, pyriproxyfen, did not affect hypocotyl elongation. Co-treatment with fenoxycarb and BR partly canceled the fenoxycarb-induced hypocotyl suppression. In addition, gene expression analysis revealed that fenoxycarb altered the BR-responsive gene expression. These results indicate that fenoxycarb is a BR action inhibitor.</p>
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Insect growth regulators with hydrazide moiety inhibit strigolactone biosynthesis in rice 査読あり
47 ( 1 ) 2022年
記述言語：英語 掲載種別：研究論文（学術雑誌）
本研究では、SLの生合成を阻害する新規リード化合物を同定するために、SLの生合成に対する農薬の影響を評価した。その結果、クロマフェノジドとメトキシフェノジドがSL生合成の新規リード阻害剤であることを示唆した。
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Canonical strigolactones are not the major determinant of tillering but important rhizospheric signals in rice 査読あり
Shinsaku Ito, Justine Braguy, Jian You Wang , Akiyoshi Yoda, Valentina Fiorilli, Ikuo Takahashi, Muhammad Jamil, Abrar Felemban, Sho Miyazaki, Teresa Mazzarella, Guan-Ting Erica Chen, Akihisa Shinozawa, Aparna Balakrishna, Lamis Berqdar, Chakravarty Rajan, Shawkat Ali, Imran Haider, Yasuyuki Sasaki, Shunsuke Yajima, Kohki Akiyama, Luisa Lanfranco, Matias D Zurbriggen, Takahito Nomura, Tadao Asami, Salim Al-Babili
Sci Adv. 8 ( 44 ) 2022年
記述言語：英語 掲載種別：研究論文（学術雑誌）
Strigolactones (SLs) are a plant hormone inhibiting shoot branching/tillering and a rhizospheric, chemical signal that triggers seed germination of the noxious root parasitic plant Striga and mediates symbiosis with beneficial arbuscular mycorrhizal fungi. Identifying specific roles of canonical and noncanonical SLs, the two SL subfamilies, is important for developing Striga-resistant cereals and for engineering plant architecture. Here, we report that rice mutants lacking canonical SLs do not show the shoot phenotypes known for SL-deficient plants, exhibiting only a delay in establishing arbuscular mycorrhizal symbiosis, but release exudates with a significantly decreased Striga seed-germinating activity. Blocking the biosynthesis of canonical SLs by TIS108, a specific enzyme inhibitor, significantly lowered Striga infestation without affecting rice growth. These results indicate that canonical SLs are not the determinant of shoot architecture and pave the way for increasing crop resistance by gene editing or chemical treatment.
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Nitric Oxide Signaling for Aerial Mycelium Formation in Streptomyces coelicolor A3(2) M145 査読あり 国際誌
Sota Honm, Shinsaku Ito, Shunsuke Yajima, Yasuyuki Sasaki
Appl Environ Microbiol . 88 ( 23 ) 2022年
担当区分：責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌）
Nitric oxide (NO) is a well-known signaling molecule in various organisms. Streptomyces undergoes complex morphological differentiation, similar to that of fungi. A recent study revealed a nitrogen oxide metabolic cycle that forms NO in Streptomyces coelicolor A3(2) M145. Further, endogenously produced NO serves as a signaling molecule. Here, we report that endogenously produced NO regulates cyclic 3',5'-diguanylate (c-di-GMP) levels and controls aerial mycelium formation through the c-di-GMP-binding transcriptional regulator BldD in S. coelicolor A3(2) M145. These observations provide important insights into the mechanisms regulating morphological differentiation. This is the first study to demonstrate a link between NO and c-di-GMP in S. coelicolor A3(2) M145. Morphological differentiation is closely linked to the initiation of secondary metabolism in actinomycetes. Thus, the NO signaling-based regulation of aerial mycelium formation has potential applications in the fermentation industry employing useful actinomycetes.
DOI： 10.1128/aem.01222-22
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Nitric Oxide Signaling for Actinorhodin Production in Streptomyces coelicolor A3(2) via the DevS/R Two-Component System 査読あり 国際誌
Sota Honm, Shinsaku Ito, Shunsuke Yajima, Yasuyuki Sasaki
Appl Environ Microbiol . 87 ( 14 ) 2021年
担当区分：責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌）
Nitric oxide (NO) is an important signaling molecule in eukaryotic and prokaryotic cells. A previous study revealed an NO synthase-independent NO production metabolic cycle in which the three nitrogen oxides, nitrate (NO3-), nitrite (NO2-), and NO, were generated in the actinobacterium Streptomyces coelicolor A3(2). NO was suggested to act as a signaling molecule, functioning as a hormone that regulates secondary metabolism. Here, we demonstrate the NO-mediated regulation of the production of the blue-pigmented antibiotic actinorhodin (ACT), via the heme-based DevS/R two-component system (TCS). Intracellular NO controls the stabilization or inactivation of DevS, depending on the NO concentration. An electrophoretic mobility shift assay and chromatin immunoprecipitation-quantitative PCR analysis revealed the direct binding between DevR and the promoter region of actII-ORF4, resulting in gene expression. Our results indicate that NO regulates the DevS/R TCS, thereby strictly controlling the secondary metabolism of S. coelicolor A3(2).
DOI： 10.1128/AEM.00480-21
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Structural basis of the conformational changes in Microbacterium hydrocarbonoxydans IclR transcription factor homolog due to ligand binding 査読あり
Tomonori Akiyama, Yasuyuki Sasaki, Shinsaku Ito, Shunsuke Yajima
Biochim Biophys Acta Proteins Proteom . 1869 ( 7 ) 2021年
記述言語：英語 掲載種別：研究論文（学術雑誌）
Microbacterium hydrocarbonoxydans has been isolated using an unnatural acylhydrazide compound as the sole carbon source. The compound is hydrolyzed by bacterial hydrazidase, and the gene expression of the enzyme is considered to be controlled by a transcription factor of the Isocitrate lyase Regulator (IclR) family, belonging to the one-component signaling systems. Recently, we reported the crystal structure of an unliganded IclR homolog from M. hydrocarbonoxydans, named putative 4-hydroxybenzoate response regulator (pHbrR), which has a unique homotetramer conformation. In this study, we report the crystal structure of pHbrR complexed with 4-hydroxybenzoic acid, the catalytic product of hydrazidase, at 2.0 Å resolution. pHbrR forms a homodimer with multimeric rearrangement in the unliganded state. Gel filtration column chromatography results suggested dimer-tetramer rearrangement. We observed conformational change in the loop region covering the ligand-binding site, and domain rearrangements in the monomer. This study reports the first liganded IclR family protein structure that demonstrates large structural rearrangements between liganded and unliganded proteins, which may represent a general model for IclRs.
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Triflumizole as a Novel Lead Compound for Strigolactone Biosynthesis Inhibitor 査読あり 国際誌
Kojiro Kawada, Yuya Uchida, Ikuo Takahashi, Takahito Nomura, Yasuyuki Sasaki, Tadao Asami, Shunsuke Yajima, Shinsaku Ito
Molecules . 25 ( 23 ) 2020年
記述言語：英語 掲載種別：研究論文（学術雑誌）
Strigolactones (SLs) are carotenoid-derived plant hormones involved in the development of various plants. SLs also stimulate seed germination of the root parasitic plants, Striga spp. and Orobanche spp., which reduce crop yield. Therefore, regulating SL biosynthesis may lessen the damage of root parasitic plants. Biosynthetic inhibitors effectively control biological processes by targeted regulation of biologically active compounds. In addition, biosynthetic inhibitors regulate endogenous levels in developmental stage- and tissue-specific manners. To date, although some chemicals have been found as SL biosynthesis inhibitor, these are derived from only three lead chemicals. In this study, to find a novel lead chemical for SL biosynthesis inhibitor, 27 nitrogen-containing heterocyclic derivatives were screened for inhibition of SL biosynthesis. Triflumizole most effectively reduced the levels of rice SL, 4-deoxyorobanchol (4DO), in root exudates. In addition, triflumizole inhibited endogenous 4DO biosynthesis in rice roots by inhibiting the enzymatic activity of Os900, a rice enzyme that converts the SL intermediate carlactone to 4DO. A Striga germination assay revealed that triflumizole-treated rice displayed a reduced level of germination stimulation for Striga. These results identify triflumizole as a novel lead compound for inhibition of SL biosynthesis.
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Structural basis of substrate recognition by the substrate binding protein (SBP) of a hydrazide transporter, obtained from Microbacterium hydrocarbonoxydans
Shimamura K, Akiyama T, Yokoyama K, Takenoya M, Ito S, Sasaki Y, Yajima S.
Biochem Biophys Res Commun . 525 ( 3 ) 2020年
記述言語：英語 掲載種別：研究論文（学術雑誌）
Microbacterium hydrocarbonoxydans was isolated, using hydrazide compounds as its sole carbon source. The key enzyme that metabolizes these compounds was identified as hydrazidase, and the operon containing the gene coding for the enzyme, was revealed by genome sequencing. The operon also contained genes coding for an ATP-binding cassette transporter (ABC transporter), which was expected to transport the hydrazide compounds. Substrate binding protein (SBP), a component subunit of the transporter, plays an important role in recognizing the correct substrates for transport. Therefore, to elucidate the mechanism of recognition of the unnatural hydrazide compounds, we determined the crystal structures of the SBP, obtained from M. hydrocarbonoxydans (Mh-SBP), complexed with and without the hydrazide compound, at 2.2 Å and 1.75 Å resolutions, respectively. The overall structures of Mh-SBP were similar to those of the SBP in oligopeptide transporters such as OppA. On comparison, the liganded and unliganded structures of Mh-SBP showed an open - close conformation change. Interestingly, the binding mode of the compound to Mh-SBP was almost identical to that of the compound to hydrazidase, suggesting that the ABC transporter served transporting these compounds. Furthermore, based on the hydrazide complex structure, paraben, the other putative substrate of the protein, was successfully used with Mh-SBP to obtain the paraben complex structure.
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Single amino acid mutation altered substrate specificity for L-glucose and inositol in scyllo-inositol dehydrogenase isolated from Paracoccus laeviglucosivorans 査読あり 国際誌
Mayu Suzuki, Kairi Koubara, Mihoko Takenoya, Kazuhiro Fukano, Shinsaku Ito, Yasuyuki Sasaki, Akira Nakamura, Shunsuke Yajima
84 ( 4 ) 2020年
記述言語：英語 掲載種別：研究論文（学術雑誌）
scyllo-inositol dehydrogenase, isolated from Paracoccus laeviglucosivorans (Pl-sIDH), exhibits a broad substrate specificity: it oxidizes scyllo- and myo-inositols as well as L-glucose, converting L-glucose to L-glucono-1,5-lactone. Based on the crystal structures previously reported, Arg178 residue, located at the entry port of the catalytic site, seemed to be important for accepting substrates. Here, we report the role of Arg178 by using an alanine-substituted mutant for kinetic analysis as well as to determine the crystal structures. The wild-type Pl-sIDH exhibits the activity for scyllo-inositol most preferably followed by myo-inositol and L-glucose. On the contrary, the R178A mutant abolished the activities for both inositols, but remained active for L-glucose to the same extent as its wild-type. Based on the crystal structures of the mutant, the side chain of Asp191 flipped out of the substrate binding site. Therefore, Arg178 is important in positioning Asp191 correctly to exert its catalytic activities.
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Structural basis for the substrate recognition of aminoglycoside 7''-phosphotransferase-Ia from Streptomyces hygroscopicus 査読あり 国際誌
Mihoko Takenoya 1, Tatsuro Shimamura 2, Ryuji Yamanaka 1, Yuya Adachi 1, Shinsaku Ito 1, Yasuyuki Sasaki 1, Akira Nakamura 3, Shunsuke Yajima 1
Acta Crystallogr F Struct Biol Commun . 75 2019年
記述言語：英語 掲載種別：研究論文（学術雑誌）
Hygromycin B (HygB) is one of the aminoglycoside antibiotics, and it is widely used as a reagent in molecular-biology experiments. Two kinases are known to inactivate HygB through phosphorylation: aminoglycoside 7''-phosphotransferase-Ia [APH(7'')-Ia] from Streptomyces hygroscopicus and aminoglycoside 4-phosphotransferase-Ia [APH(4)-Ia] from Escherichia coli. They phosphorylate the hydroxyl groups at positions 7'' and 4 of the HygB molecule, respectively. Previously, the crystal structure of APH(4)-Ia was reported as a ternary complex with HygB and 5'-adenylyl-β,γ-imidodiphosphate (AMP-PNP). To investigate the differences in the substrate-recognition mechanism between APH(7'')-Ia and APH(4)-Ia, the crystal structure of APH(7'')-Ia complexed with HygB is reported. The overall structure of APH(7'')-Ia is similar to those of other aminoglycoside phosphotransferases, including APH(4)-Ia, and consists of an N-terminal lobe (N-lobe) and a C-terminal lobe (C-lobe). The latter also comprises a core and a helical domain. Accordingly, the APH(7'')-Ia and APH(4)-Ia structures fit globally when the structures are superposed at three catalytically important conserved residues, His, Asp and Asn, in the Brenner motif, which is conserved in aminoglycoside phosphotransferases as well as in eukaryotic protein kinases. On the other hand, the phosphorylated hydroxyl groups of HygB in both structures come close to the Asp residue, and the HygB molecules in each structure lie in opposite directions. These molecules were held by the helical domain in the C-lobe, which exhibited structural differences between the two kinases. Furthermore, based on the crystal structures of APH(7'')-Ia and APH(4)-Ia, some mutated residues in their thermostable mutants reported previously were located at the same positions in the two enzymes.
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Synthesis and Biological Evaluation of Novel Triazole Derivatives as Strigolactone Biosynthesis Inhibitors 査読あり 国際誌
Kojiro Kawada, Ikuo Takahashi, Minori Arai, Yasuyuki Sasaki, Tadao Asami, Shunsuke Yajima, Shinsaku Ito
J Agric Food Chem . 67 ( 22 ) 2019年
記述言語：英語 掲載種別：研究論文（学術雑誌）
Strigolactones (SLs) are one of the plant hormones that control several important agronomic traits, such as shoot branching, leaf senescence, and stress tolerance. Manipulation of the SL biosynthesis can increase the crop yield. We previously reported that a triazole derivative, TIS108, inhibits SL biosynthesis. In this study, we synthesized a number of novel TIS108 derivatives. Structure-activity relationship studies revealed that 4-(2-phenoxyethoxy)-1-phenyl-2-(1 H-1,2,4-triazol-1-yl)butan-1-one (KK5) inhibits the level of 4-deoxyorobanchol in roots more strongly than TIS108. We further found that KK5-treated Arabidopsis showed increased branching phenotype with the upregulated gene expression of AtMAX3 and AtMAX4. These results indicate that KK5 is a specific SL biosynthesis inhibitor in rice and Arabidopsis.
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Structural basis of L-glucose oxidation by scyllo-inositol dehydrogenase: Implication for a novel enzyme subfamily classification 査読あり
Fukano K, Ozawa K, Kokubu M, Shimizu T, Ito S, Sasaki Y, Nakamura A, Yajima S
PLOS ONE 13 ( 51 ) e0198010 2018年05月
記述言語：英語 掲載種別：研究論文（学術雑誌）
非天然化合物であるL-glucoseを唯一の炭素源として生育可能な微生物より単離したL-glucose dehydrogenaseの構造を解析し、その基質認識機構をあきらかとした。それに加え、新規のサブファミリィーを構成することを明らかとした。
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Nitrate analogs as attractants for soybean cyst nematode. 査読あり
Hosoi A, Katsuyama T, Sasaki Y, Kondo T, Yajima S, Ito S.
Bioscience, Biotechnology, and Biochemistry 81 ( 8 ) 1542 - 1547 2017年08月
記述言語：英語 掲載種別：研究論文（学術雑誌）
ダイズシストセンチュウが硝酸イオンやそのアナログに誘引されることを見出した。
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Regulation of Strigolactone Biosynthesis by Gibberellin Signaling. 査読あり
Ito S, Yamagami D, Umehara M, Hanada A, Yoshida S, Sasaki Y, Yajima S, Kyozuka J, Ueguchi-Tanaka M, Matsuoka M, Shirasu K, Yamaguchi S, Asami T.
Plant Physiology 174 1250 - 1259 2017年06月
記述言語：英語 掲載種別：研究論文（学術雑誌）
ストリゴラクトンの生合成がジベレリンシグナルによって制御されることを見出し、両ホルモン間のクロストークの存在を明らかとした。
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Crystal structure of an IclR homologue from Microbacterium sp. strain HM58-2 査読あり
Tomonori Akiyama, Yusuke Yamada, Naoki Takaya, Shinsaku Ito, Yasuyuki Sasaki and Shunsuke Yajima
Acta Crystallogr Sect F Struct Biol Cryst F73 16 - 23 2017年
記述言語：英語 掲載種別：研究論文（学術雑誌）
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Structural basis of the substrate recognition of hydrazidase isolated from Microbacterium sp. strain HM58-2, which catalyzes acylhydrazide compounds as its sole carbon source 査読あり
Tomonori Akiyama, Misaki Ishii, Atsushi Takuwa, Ken-Ichi Oinuma, Yasuyuki Sasaki, Naoki Takaya, Shunsuke Yajima
Biochemical and Biophysical Research Communications 482 1007 - 1012 2017年
記述言語：英語 掲載種別：研究論文（学術雑誌）
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A role of nitrite reductase (NirBD) for NO homeostatic regulation in Streptomyces coelicolor A3(2) 査読あり
Yuriya Yukioka; Tsukiko Tanahashi; Keisuke Shida; Haruka Oguchi; Shota Ogawa; Chiaki Saito; Shunsuke Yajima; Shinsaku Ito; Kanju Ohsawa; Hirofumi Shoun; Yasuyuki Sasaki
FEMS Microbiology Letters 2016年10月
担当区分：責任著者 記述言語：英語 掲載種別：研究論文（学術雑誌）
放線菌に見出した新規なシグナリングシステムの制御機構に、亜硝酸還元酵素が関与し得ていることを見出した。
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1,10-Phenanthroline and its derivatives are novel hatching stimulants for soybean cyst nematodes. 査読あり
Nonaka S, Katsuyama T, Kondo T, Sasaki Y, Asami T, Yajima S, Ito S
Bioorg Med Chem Lett. 2016年09月
記述言語：英語 掲載種別：研究論文（学術雑誌）
ダイズシストセンチュウに対して、新規な孵化誘導物質を発見し、解析した。